sign in sign up
<<
Home , Protein purification

A BEGINNER’S GUIDE TO

by Deborah Grainger

Starting Immunohistochemistry experiments for the first time? Comfortable with the method, but searching for few extra tips? Guest Blogger Sarah Etheridge talks about her experiences of IHC experiments in the form of ten handy hints…

Immunohistochemistry, or IHC, allows you to visualize in intact tissue retaining its microstructure, or as I like to say: ‘real life’. One of the advantages of this ‘real life’ visualization is that it allows for comparison between healthy and diseased tissues, thus the application is invaluable to both scientist and pathologist alike. The IHC protocol is straightforward, but contains many steps that may require initial optimization to ensure specific binding and optimal visualization of the target . Here, I outline some tips for each of these steps and highlight which may need a little tweaking.

1. Tissue preparation Tissue samples can be frozen or fixed. Freezing the sections generally maintains the conformation of the target allowing superior antibody binding, but small ice crystals may form in the tissue [1, 2] and these sections may not be a good choice for long-term storage. Fixed and embedded tissue is a better alternative if you wish to hang on to your slides a little longer. The most common method of tissue fixation and embedding is formaldehyde fixation with paraffin embedding (FFPE). (It is possible to store FFPE embedded biopsies indefinitely at room temperature – making them an important resource for historical studies in medicine!).

For most proteins, it is a good IHC viewed on a laser scanning idea to add a permeabilization confocal microscope. Blue color: UV-range step to your protocol. autofluorescence from postganglionic neurons. Source: Wikimedia commons.

2. Tissue Preparation: Other things to try Fixing tissue sections, before staining, with ice cold 50:50 methanol-acetone (MeAc) or 4% paraformaldehyde (PFA) may enhance antibody binding. MeAc, for example, dissolves cytoplasmic proteins allowing better visualization of membrane bound proteins.

3. Antigen retrieval Formaldehyde fixing causes cross-linking of proteins within the tissue, maintaining tissue morphology but denaturing the epitopes recognized by the antibody [3]. Therefore, antigen retrieval is usually carried out before staining FFPE sections to unmask hidden or denatured target epitopes. This can be heat-induced A BEGINNER’S GUIDE TO IMMUNOHISTOCHEMISTRY

or enzymatic [1, 4] with factors such as temperature, pH and time affecting antigen retrieval. It is a good idea to test a ‘battery’ or panel of antigen retrieval methods for each new antibody and tissue combination to determine optimal staining and rule out any non-specific background staining [5]. A good starting point is boiling in citrate buffer at pH 6.0 in the microwave. Antigen retrieval is not needed for frozen sections, but the sections should be air dried for at least an hour prior to staining. De-waxing of FFPE sections in xylene and alcohol must be carried out prior to antigen retrieval and staining; following de-waxing, sections are rehydrated in decreasing levels of alcohol and then water, which leads me to my next top tip…

4. Sample handling …Once sections are de-waxed and rehydrated it is important not to let them dry out! So use a specialized IHC staining tray to keep them in during incubations – many vendors sell these, they help keep tissue sections in a humidified environment. Alternatively, use a tray with tissue soaked in water. Top tip: Use a PAP pen– a special marking pen that provides a thin film-like hydrophobic barrier when drawn around a specimen – to keep solutions close to the sections once the permeabilization step has been carried out. Its use is not entirely essential but can be useful. (Don’t add PAP pen before permeabilization or MeAc fixation as it may be dissolved by Triton or MeAc and will bind to your tissue.)

5. Permeabilization For most proteins, it is a good idea to add a permeabilization step to your protocol. Permeabilization may not be necessary for transmembrane proteins whose epitopes are in the extracellular region. Permeabilization involves incubation with a (e.g. 0.1% Triton-X100 in PBS). can also act as surfactants, breaking through some of the protein cross-linking that occurs during formaldehyde fixing, therefore aiding antibody binding to the correct epitope [1] and reducing non-specific hydrophobic interactions.

Top tip: If staining isn’t working, try including detergent such as Triton at a lower level in all solutions (particularly for FFPE staining).

6. Blocking The blocking solution binds to non-specific binding sites within the tissue, [1] thus preventing non-specific binding of the primary and secondary to tissue components. Sections are incubated with an ‘innocuous protein solution’ such as bovine serum albumin (BSA) or serum from the host of the secondary antibody – I usually start with 3% BSA solution for 20-30 minutes. Proteintech also routinely uses 3% BSA, but can also use 5-10% goat serum as an alternative (as the secondary antibody it employs is raised in goat).

7. Choosing your primary antibody Your choice of primary antibody should depend on factors such as expression levels of the protein- of-interest and any other antibodies used in the case of co-localization. Antibodies from different Immunohistochemistry of formalin fixed, paraffin- species must be used in the case of the latter so embedded cervical cancer using Proteintech’s that different secondary antibodies with unique KRT13 polyclonal antibody 10085-376 (viewed conjugates can be used to distinguish between under a 40x lens). Polyclonal antibodies generally them. You must also choose whether a monoclonal produce a brighter IHC signal as they recognize or polyclonal is more suited to your needs; several epitopes on the same protein. Source: monoclonal antibodies give a highly specific signal Proteintech’s 10085-376 datasheet. whereas polyclonal antibodies can give a brighter one, particularly if expression levels of the protein of interest are low. A BEGINNER’S GUIDE TO IMMUNOHISTOCHEMISTRY

Box 1: Antibody controls There is a chance of cross-reactivity and non-specific Western blotting is used to check that the antibody is binding of other antibodies in the host serum. Purification detecting a protein of the right size. Multiple bands on a techniques such as /G purification, affinity suggest non-specific antibody binding. purification or pre-absorption are used to prevent this from occurring. Proteintech affinity purifies all its Antibody specificity can be checked by pre-incubation of antibodies with target antigen during product preparation; the primary antibody with the antigenic peptide (an however, if you wish to carry out controls to test the adsorption test). This step is not necessary if the antibody specificity of primary antibody binding, a few options are is affinity-purified, as the specific peptide sequence is set out below: used for the protein purification [6].

The ideal control would be to use tissue that does not If the antibody is custom made, carry out Western blotting contain the protein of interest, for example: tissue from with serum from the various steps of the production a knock-out mouse or cells where the target protein is process to determine whether the band representing your knocked down i.e. with siRNA protein is detected by the correct fractions.

8. Antibody dilutions A guide for dilutions is usually provided with the antibody sheet, otherwise dilutions of 1 in 50 – 1 in 300 are good starting points for optimization; perhaps try a panel of primary antibody dilutions with a brand new antibody. Dilute the primary antibody in blocking solution (e.g. 3% BSA in PBS), then incubate for 1-2 hours at room temperature, or overnight at 39°F/4°C.

9. Washing Wash off the primary antibody with wash solution such as PBS. A quick wash followed by three 5 minute washes should be sufficient. Frozen sections can be quite fragile, so drop solution slowly next to the section e.g. with a 1 ml pipette. Top tip: Alter the washing buffer to Tris-buffered Saline (TBS) rather than PBS

Box 2: Detection Detection of the primary antibody is usually carried Keep sections in the dark once fluorescent antibodies out with a secondary antibody directed against have been added to prevent bleaching. immunoglobulins of the host species of the primary antibody, conjugated to a fluorescent (e.g. FITC) or After incubation, wash the sections several times with a chromogenic enzymatic tag (such as horse-radish PBS before adding mounting medium to the sections and peroxidase (HRP). Follow these tips for happy detecting! carefully lowering a coverslip over the top.

Background staining can vary between experiments, so it Lower the coverslip gently to avoid air bubbles. Take is a good idea to perform a negative control in which the particular care with frozen sections, try not to squeeze primary antibody is omitted for each experiment. or press the coverslip too hard as this may disrupt the tissue. Dilute the secondary antibody in blocking solution, usually at around 1 in 800 – 1 in 1000 dilution (although It is often useful to stain the nuclei e.g. with DAPI, which this can be adjusted in future). can be included in a wash step or in the mounting medium. For fluorescence experiments: It is possible to use primary antibodies directly Check that the fluorophore you intend to use is detected conjugated to fluorescent tags. This saves time and by your microscope. It is important to avoid spectral reduces false signal but the level of signal will be lower. overlap of the fluorochromes tagged to the secondary There are methods of amplifying low-level signals, antibody, cross-reactivity between secondary antibodies including avidin-biotin-complex (ABC) and labelled and bleed-through of the signal whilst imaging. streptavidin-biotin method (LSAB). A BEGINNER’S GUIDE TO IMMUNOHISTOCHEMISTRY

10. Suggested starting points for optimization Don’t be intimidated by the many facets of an IHC protocol that could need optimization. Here’s a list of the first steps you should consider optimizing in the first stages of your IHC experiments: Antigen retrieval for FFPE sections Primary antibody dilution Blocking solution

References 1. Daneshtalab N, Doré JJ and Smeda JS, Troubleshooting tissue specificity and antibody selection: Procedures in immunohistochemical studies., J Pharmacol Toxicol Methods., 2010;61(2):127-35. 2. Shi SR, Liu C, Pootrakul L et al., Evaluation of the value of frozen tissue section used as “gold standard” for immunohistochemistry., Am J Clin Pathol., 2008;129(3):358-66. 3. Fox CH, Johnson FB, Whiting J et al., Formaldehyde fixation. J Histochem Cytochem., 1985;33:845–853. 4. Yamashita S et al., Heat-induced antigen retrieval: mechanisms and application to histochemistry.Prog Histochem Cytochem., 2007;41:141-200. 5. Shi SR, Cote RJ and Taylor CR, Antigen retrieval immunohistochemistry: past, present, and future. J Histochem Cytochem., 1997;45(3):327-43. 6. Saper CB and Sawchenko PE, Magic peptides, magic antibodies: guidelines for appropriate controls for immunohistochemistry. J Comp Neurol., 2003;465:161–163.

0615 Lit. No. 161292W